Therapies which act on neuropeptide S receptors

ABSTRACT

Compositions and methods that act on Neuropeptide S receptors (NPSR) (also known as “TGR23” or “vasopressin receptor-related receptor 1 (VRR1)”) to cause desired effects in the bodies of human or animal subjects. Neuropeptide S (NPS) and other agonists of the NPSR may be administered to cause arousal, awakening, alertness, spontaneous movement, bronchoconstriction, contraction of bronchial smooth muscle or other effects. Antagonists of the NPSR may be administered to cause decreased arousal, decreased awakening, decreased alertness, decreased spontaneous movement, sleep, somnolence, sedation, anxiolytic effects, normalized sleep patterns, normalized sleep stages, increased duration of sleep, bronchodilation, relaxation of broncheal smooth muscle or other effects.

RELATED APPLICATION

This application is a Section 371 national stage of PCT InternationalPatent Application No. PCT/US05/14312 filed Apr. 25, 2005 which claimspriority to U.S. Provisional Patent Application No. 60/565,269 filed onApr. 23, 2004, the entirety of which is expressly incorporated herein byreference.

STATEMENT REGARDING GOVERNMENT SUPPORT

This invention was made with Government support under Grant Nos.MH-60231 awarded by the National Institutes of Health. The Governmenthas certain rights in this invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 15, 2014, isnamed UCIVN-064US SL-FILED.txt and is 6,395 bytes in size.

FIELD OF THE INVENTION

The present invention relates generally to the fields of biology andmedicine, and more particularly to compositions and methods foraffecting neuropeptide S (NPS) receptors to treat various disorders,including but not limited to narcolepsy, insomnia, drowsiness,hypersomnia, anxiety, asthma and allergies.

BACKGROUND OF THE INVENTION

As described in United States Provisional Patent Application No.60/565,269, an endogenous brain protein, Neuropeptide S (NPS) which isbelieved to affect arousal, wakefulness, propensity for movement, asthmaand some allergic responses, stress associated with several anxietydisorders and other physiological functions. Human NPS (hNPS) has thefollowing amino acid sequence:Ser-Phe-Arg-Asn-Gly-Val-Gly-Thr-Gly-Met-Lys-Lys-Thr-Ser-Phe-Gln-Arg-Ala-Lys-Ser-OH(SEQ ID NO:1). It is now available commercially as product No. E010051from PentaBiotech, Inc., Union City, Calif.

Neuropeptide S is the endogenous ligand for the Neuropeptide S receptor(NPSR), which has also been referred to as TGR23 and vasopressinreceptor-related receptor 1 (VRR1) (Genbank accession no. BD183774,BD183814, BD183773). The NPSR is a G protein coupled receptor (GPCR).

NPS acts as an agonist of the NPSR, causing dose dependent intracellularCa++ mobilization as well as adenyl cyclase accumulation measured bycAMP assay. NPS is also involved in G protein-coupled receptor forasthma susceptibility. Y. L. Xu, R. X. Reinscheid, S. Huitron-Resendiz,S. D. Clark, Z. Wang, S. H. Lin, F. A. Brucher, J. Zeng, N. K. Ly, S. J.Henriksen, L. d'Lecea, O. Civelli, Neuron, 43, 487-497 (2004).

PCT International Patent Publication WO 02/31145 A1 (Sato) describescertain functions of NPS and the NPSR. However, PCT International PatentPublication WO 02/31145 A1 (Sato) does not describe specificpharmacological characteristics and physiological functions of the GPCRsystem, NPS, the NPSR or its amino acid sequence (Genbank accession no.BD183774, BD183814, BD183773). PCT International Patent Publication WO02/31145 A1 (Sato) is expressly incorporated herein in its entirety.

PCT International Patent Publication WO 2005/021555 A1 (TakedaPharmaceutical Company) described certain bicyclic piperazine compoundsthat a NPSR antognists and are purportedly useable in the prevention andtreatment of certain cancers. PCT International Patent Publication WO2005/021555 A1 (Takeda Pharmaceutical Company) is expressly incorporatedherein by reference.

Definitions

When used in this patent application, the following terms andabbreviations shall be interpreted as follows:

The verb “to treat” and formatives/tenses thereof (i.e., treats,treating, treatment, etc.) shall include therapeutic, preventative,paliative, experimental and diagnostic treatments.

The term “subject” shall include human and other animal patients andnon-patient subjects who receive therapeutic, preventative, experimentalor diagnostic treatment, humans and animals who have a disease or arepredisposed to a disease and/or laboratory animals or humans who on whomtests or experiments are performed.

Compositions or methods “comprising” one or more recited elements mayinclude other elements not specifically recited. For example, acomposition that comprises NPS may encompass both an isolated NPS ofhuman or animal origin as a component of a larger polypeptide sequenceor as part of a composition or preparation that includes otherpharmacologically active or inactive ingredients or components.

The term “Isolated” means purified, substantially purified or partiallypurified. Isolated can also mean present in an environment other than anaturally occurring environment. For example, NPS that is not present inor mixed with nervous tissue, extracellular fluid, cerebrospinal fluidof other body fluids/tissues in which NPS would ordinarily be found whennaturally occurring shall be deemed to be isolated NPS.

The verb “to administer” and formatives/tenses thereof (i.e.,administers, administering, administration, etc.) shall include any actof providing or delivering a substance to a human or animal subject orto in vitro preparation, including but not limited to oral, enteral,intravenous, intraarterial, parenteral, subcutaneous, intradermal,transdermal, transmucosal, buccal, sublingual, lingual, rectal,intraperitoneal, central, intraventricular, intrathecal, epidural,spinal, topical, alveolar, inhalational, transtracheal and all otherroutes by which such substance may be administered or provided.Additionally, acts that cause, induce, stimulate, accelerate, enhance orfacilitate the biosynthesis or endogenous production of an endogenousnaturally occurring substance (e.g., endogenous NPS) shall also beconstrued as acts of “administering” such substance. Additionally, actsthat inhibit, decrease, slow, interfere with or block the metabolismand/or clearance of a naturally occurring substance (e.g., endogenousNPS) so as to result in the presence of increased or greater amounts ofthe substance within the body of a human or animal subject shall also beconstrued as acts of “administering” such substance.

The acronym “NPS” shall mean “neuropeptide S”, including those of humanand animal origin having the amino acid sequences shown in FIG. 1.

The acronym “hNPS” shall mean “human neuroeptide S”, the amino acidsequence of which is:Ser-Phe-Arg-Asn-Gly-Val-Gly-Thr-Gly-Met-Lys-Lys-Thr-Ser-Phe-Gln-Arg-Ala-Lys-Ser-OH(SEQ ID NO: 1).

The acronym “NPSR” shall mean “neuropeptide S receptor” or “TGR23” or“vasopressin receptor-related receptor 1.”

The acronym “WT” shall mean “wildtype.”

The acronym “GPCR” shall mean “G protein-coupled receptor.”

The acronym “SNP” shall mean “single nucleotide polymorphism.”

The acronym “MAPK” shall mean “mitogen-activated protein kinase.”

The acronym “LC” shall mean “locus coeruleus.”

The acronym “Ala” or the symbol “A” shall mean “Alanine.”

The acronym “Arg” or the symbol “R” shall mean “Arginine.”

The acronym “Asn” or the symbol “N” shall mean “Asparagine.”

The acronym “Asp” or the symbol “D” shall mean “Aspartic Acid.”

The acronym “Asx” or the symbol “B” shall mean “Asparagine or AsparticAcid.”

The acronym “Cys” or the symbol “C” shall mean “Cystine.”

The acronym “Gln” or the symbol “Q” shall mean “Glutamine.”

The acronym “Glu” or the symbol “E” shall mean “Glutamic Acid.”

The acronym “Glx” or the symbol “Z” shall mean “Glutamine or GlutamicAcid.”

The acronym “Gly” or the symbol “G” shall mean “Glycine.”

The acronym “His” or the symbol “H” shall mean “Histidine.”

The acronym “Ile” or the symbol “I” shall mean “Isoleucine.”

The acronym “Leu” or the symbol “L” shall mean “Leucine.”

The acronym “Lys” or the symbol “K” shall mean “Lysine.”

The acronym “Met” or the symbol “M” shall mean “Methionine.”

The acronym “Phe” or the symbol “F” shall mean “Phenylalanine.”

The acronym “Pro” or the symbol “P” shall mean “Proline.”

The acronym “Ser” or the symbol “S” shall mean “Serine.”

The acronym “Thr” or the symbol “T” shall mean “Threonine.”

The acronym “Trp” or the symbol “W” shall mean “Tryptophan.”

The acronym “Tyr” or the symbol “Y” shall mean “Tyrosine.”

The acronym “Val” or the symbol “V” shall mean “Valine.”Additionalterms, acronyms and/or symbols are defined elsewhere in this patentapplication.

SUMMARY OF THE INVENTION

The present invention provides a composition of matter comprisingisolated NPS.

Further in accordance with the present invention, NPSR agonists may beadministered to human or veterinary subjects in effective dosages and byeffective routes of administration to cause arousal, awakening,alertness, anxiolytic effects, spontaneous movement or to inducebronchoconstriction, bronchial smooth muscle contraction or asthma(e.g., for diagnostic or experimental purposes) and/or other effects asdescribed herein. Thus, NPSR agonists may be useable to treat disorderssuch as; narcolepsy, hypersomnia, lack of alertness, lack ofattentiveness, absentmindedness, lack of or aversion to movement orexercise, anxiety, stress and stress related disorders, and otherdisorders as described herein. Examples of NPSR agonists that may beadministered in accordance with this invention include but are notnecessarily limited to NPS, isolated NPS, fragments of NPS, compositionsthat comprise NPS or other agonists of the NPSR.

Still further in accordance with the present invention, NPSR antagonistsand preparations that comprise NPSR antagonists may be administered tohuman or veterinary subjects in effective dosages and by effectiveroutes of administration to cause; decreased arousal, decreasedawakening, decreased alertness, decreased spontaneous movement, sleep,somnolence, sedation, normalized sleep patterns, normalized sleepstages, increased duration of sleep, bronchodilation, relaxation ofbroncheal smooth muscle and/or other effects as described herein. Thus,NPSR antagonists (e.g., compounds of General Formula I above) andpreparations that comprise NPSR antagonists may useable to treatdisorders such as insomnia, sleep disorders, decreased duration of sleepor frequent awakening, disorders that cause excessive spontaneousmovement, some behavioral disorders, bronchitis, obstructive pulmonarydisease, asthma, allergic conditions and other disorders as describedherein. Examples of NPSR antognists that may be administered inaccordance with this invention include but are not necessarily limitedto those of General Formula I, as follows:

-   -   wherein R¹ comprises acyl; R² comprises an optionally        substituted hydrocarbon group; R³ comprises an optionally        substituted hydrocarbon group; R⁴ comprises an optionally        substituted hydrocarbon group; n is 0 to 4; and X comprises        oxygen, sulfur, etc.) or a salt of such compound.

Still further aspects and objects of the present invention may beunderstood from the detailed description and examples set forthherebelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows primary structures of NPS from human, chimpanzee, rat,mouse, dog and chicken. Amino acids divergent from the human sequenceare shown in bold type. Sequences were deduced from Genbank entriesBD168686 (human), BD168712 (rat), BD168690 (mouse), BU293859 (chicken),and genome sequencing traces 231487919 (chimpanzee) and 250468833 (dog).

FIGS. 2A-2C are graphs showing pharmacological characterization of thehuman NPSR. FIG. 2A is a dose response curve of [Ca²⁺]i mobilizationinduced by human, rat and mouse NPS in an HEK cell line stablyexpressing human NPSR. FIG. 2B shows saturation binding of [¹²⁵I]Y¹⁰-NPS (4 pM-1.7 nM) to CHO cells stably expressing human NPSR. FIG. 2Cshows displacement of 0.15 nM [¹²⁵I] Y¹⁰-NPS by increasingconcentrations of unlabeled human NPS. Data from triplicate experimentsare shown as means±SEM.

FIG. 3A and 3B are bar graphs showing tissue distribution of NPSprecursor (FIG. 3A) and NPSR mRNA (FIG. 3B) in rat tissues. QuantitativeRT-PCR was used to measure transcript levels of NPS precursor (left) andNPSR mRNA (right) in 45 rat tissues. Transcript levels were normalizedto β-actin. pbl, peripheral blood leucocytes.

FIGS. 4A-4H show expression of NPS precursor mRNA in the pontine area ofthe rat brain. FIG. 4A is a schematic drawing of a cross section of thepontine area of the rat brain. FIG. 4B is an autoradiogram of NPS mRNAexpression in LC area. FIGS. 4C-4E are dark field images of double insitu hybridization of NPS precursor mRNA (white) and TH mRNA (dark blue)in LC area. FIG. 4D is a higher magnification view of area 4D-4D of FIG.4C. FIG. 4E is a higher magnification view of a more caudal section.FIGS. 4F-4H are dark field images of double in situ hybridization of NPSprecursor mRNA (white) and CRF mRNA (dark blue) at mid-level of LC area(F) and rostral LC (G). (H) Higher magnification of the area indicatedby an arrow in (G). TH, tyrosine hydroxylase; NPS, neuropeptide S; CRF,corticotropin-releasing factor; Landmarks: Cb, cerebellum; 4V, 4thventricle. Scale bar is 500 μm in (C), 250 μm in all other pictures.

FIGS. 5A-5I show the distribution of NPS precursor mRNA expression inrat brain. FIGS. 5A, 5D and 5G are drawings and FIGS. 4B, 4C, 4E, 4F, 4Hand 4I are autoradiograms of sections of the rat brains. (4B and4C-(Bregma −9.68 mm), (4E and 4F-Bregma −2.80 mm) and (4H and 4I-Bregma−3.14 mm), respectively (Paxinos and Watson, 1997). (B), (C), (E), (F),(H), (I) Darkfield images of NPS precursor mRNA expression in coronalsections of rat brain. (E), (H) Expression of NPS precursor mRNA inboxed regions in (D) and (G), respectively. (C), (F), (I) Highermagnification of the area indicated by an arrow in (B), (E) and (H),respectively. Arrows in (F) and (I) indicate single cells showinghybridization signals for NPS precursor mRNA. LPB, lateral parabrachialnucleus; Pr5, principle sensory 5 nucleus; DMH, dorsomedial hypothalamicnucleus; Amg, amygdala. Landmarks: Cb, cerebellum; 3V, third ventricle;opt, optic tract. Scale bar is 500 μm.

FIGS. 6A-6O show the distribution of NPSR mRNA expression in rat brain.(A), (D), (G), (J) Schematic drawings of the sections shown in (B) and(C) (Bregma, 3.20 mm), (E) and (F) (Bregma −1.80 mm), (H) and (I)(Bregma −2.80 mm), (K) and (L) (Bregma −4.52 mm), respectively (Paxinosand Watson, 1997). (B), (E), (H), (K) Autoradiograms of NPSR mRNAexpression in coronal rat brain sections. Arrows in panel (B), (E), (H)and (K) indicate endopiriform nucleus (En). Arrowheads in (E), (H) and(K) refer to secondary motor cortex (M2), retrosplenial agranular cortex(RSA)/M2 and RSA, respectively. (C), (F), (I) Dark field images of boxedregions in (B), (E) and (H), respectively. (L) Dark field image ofmidline thalamic regions of section (K). (M), (N) Dark field image ofcortical regions in section (E). Arrows in (N) indicate scattered cellsexpressing NPSR mRNA in somatosensory cortex. (O) Dark field image ofcortical and subicular regions in section (K). AON, anterior olfactorynucleus; DEn: dorsal endopiriform nucleus, CM, central medial thalamicnucleus; IAM, interanteromedial thalamic nucleus, Rh, rhomboid thalamicnucleus; Re, reuniens thalamic nucleus; Amg, amygdala; Hyp,hypothalamus; S, subiculum; Prc, precommissural nucleus; PVP,paraventricular thalamus nucleus, posterior; PH, posterior hypothalamus.Landmarks: aca, anterior commissure, anterior part; pt, paratenialthalamic nuclei; opt, optic tract; D3V, dorsal 3rd ventricle; 3V, 3rdventricle; Hip, hippocampus. Scale bar is 500 μm.

FIGS. 7A and 7B show the effects of central administration of NPSproduces behavioral arousal and wakefulness. (A) Hyperlocomotion effectsof NPS in naïve and habituated mice. Naive mice were new to the testchamber while habituated animals were acclimatized for one hour prior tothe injection. In naive mice, 0.1 and 1 nmole NPS induce significanthyperlocomotion [F_(3,324)=92.83, p<0.0001, two-way ANOVA for repeatedmeasures]. The same doses of NPS also produced significant effects inhabituated animals [F_(3,336)=135.59, p<0.0001]. (B) Arousal promotingeffects of NPS in rats. NPS increases the amount of wakefulness anddecreases SWS1, SWS2 and REM sleep in rats (n=8 for each dose). **p<0.01, 0.1 nmole and 1.0 nmole compared with saline; * p <0.01, 1.0nmole compared with saline (ANOVA followed by Scheffe's post hoc test).

FIGS. 8A-8D are graphs showing the anxiolytic-like effects of NPS inmice. NPS produces dose-dependent anxiolytic- like effects in C57Bl/6mice exposed to the open field (A), light-dark box (B), elevatedplus-maze (C) and marble-burying paradigm (D). Doses and groups: alldoses are in nmole per animal; open field (n8 for each dose); light-darkbox (PBS, n=10; 0.01 nmole, n=5; 0.03 nmole, n=5; 0.1 nmole, n=5, 0.3nmole, n=11; 1 nmole, n=5; 3 nmole, n=8); elevated plus-maze (n=5 forall doses); marble burying (PBS and 0.01 nmole, n=10; 0.1 and 1 nmole,n=9). ** p<0.01, * p<0.05 compared to PBS control, ANOVA followed byDunneft's test for multiple comparisons. All data are presented asmeans±SEM.

FIG. 9 is a table showing EC₅₀ values (nM, +/− SEM) of NPS peptides andNPS fragments at two NPSR isoforms.

FIGS. 10A and 10B are graphs showing pharmacological activity of NPS atNPSR WT and NPSR Ile¹⁰⁷. A, dose-response curves of NPS to elicitmobilization of intracellular Ca²⁺ in HEK 293 cells stably expressingeither NPSR WT or NPSR Ile¹⁰⁷. NPS displays greater agonist potency atNPSR Ile¹⁰⁷. Incubations were performed in triplicate and repeated threetimes. B, Scatter-plot of logarithmic EC₅₀ values of individual HEK 293clones expressing either NPSR WT or NPSR Ile¹⁰⁷. Dose-response curvesfor each single clone were established measuring either mobilization ofintracellular Ca²⁺ (left) or induction of luciferase reporter genetranscription (right). Horizontal bars indicate mean EC₅₀ values.

FIGS. 11A and 11B are graphs showing pharmacological profile of NPSRvariants stimulating the cAMP pathway. A, dose-response curve of NPS tostimulate cAMP formation in two individual HEK 293 clones expressingeither NPSR WT or NPSR Ile¹⁰⁷. NPS displays greater agonist potency atNPSR Ile¹⁰⁷. Incubations were performed in triplicate and repeated threetimes. B, dose-response curves of NPS stimulating CRE-mediatedluciferase reporter gene expression. HEK 293 cells were transientlytransfected with plasmids containing either NPSR WT, NPSR Ile¹⁰⁷ or NPSRC-alt cDNA constructs. Bioluminescence was determined from triplicateincubations repeated at least twice.

FIGS. 12A and 12B NPS effect on cell proliferation and MAPKphosphorylation. A, NPS-induced stimulation of [³H]-thymidineincorporation in Colo205 human colon cancer cells. NPS produces adose-dependent stimulation of cell proliferation. 500 nM PGE₂ was usedas a positive control. All incubations were performed in triplicates andexperiments were repeated twice. ** p<0.01 vs. buffer control. B,stimulation of MAPK phosphorylation by increasing concentrations of NPS.Values were normalized to levels of phospho-MAPK produced by incubationwith 1 μM NPS (=100%). Phospho-MAPK was quantified by densitometricscanning of Western Blots as described under “Materials and Methods”.Assays were performed in duplicate and repeated twice.

FIGS. 13A and 13B are graphs showing structure-activity relationships ofNPS peptides and truncated NPS fragments at human NPSR variants. Ca²⁺mobilization elicited by human (h), mouse (m) or rat (r) NPS and variousNPS fragments was determined in two individual HEK 293 clones expressingeither NPSR WT (13At) or NPSR Ile¹⁰⁷ (13B). Dose-response curves werecalculated from triplicate incubations and all assays were repeated atleast twice. See Table 1 for comparison of EC₅₀ values and peptidesequences.

DETAILED DESCRIPTION AND EXAMPLES

The amino acid sequences of NPS in humans, chimpanzees, mice, rats, dogsand chickens are shown in FIG. 1. The NPSR is expressed in certainregions of the brain known to be involved in anxiety (e.g., theamygdala, thalamus and hypothalamic regions). Administration of NPS torodents can cause increased locomotion and anxiolytic effects. Also,Applicants have determined that NPS plays a roll in asthma (e.g.,constriction and/or dilation of bronchi and/or contraction/relaxation ofbronchiolar smooth muscle).

Arousal and anxiety are behavioral responses that involve complexneurocircuitries and multiple neurochemical components. As describedherein, NPS may be useable to modulate wakefulness and could alsoregulate anxiety. NPS acts by activating its cognate receptor (NPSR) andinducing mobilization of intracellular Ca²⁺. The NPSR mRNA is widelydistributed in the brain including the amygdala and the midline thalamicnuclei. Central administration of NPS increases locomotor activity inmice and decreases paradoxical (REM) sleep and slow wave sleep in rats.NPS was further shown to produce anxiolytic-like effects in mice exposedto four different stressful paradigms. Interestingly, NPS is expressedin a previously undefined cluster of cells located between the LC andBarrington's nucleus. These results indicate that NPS could be a newmodulator of arousal and anxiety. They also show that the LC regionencompasses distinct nuclei expressing different arousal-promotingneurotransmitters.

Sleep disorders and anxiety affect millions of people. Identifying andunderstanding the molecular regulators and neurocircuitries that areinvolved in sleep/wake cycles or arousal and anxious states are keys tothe development of therapeutic targets for these diseases.Neurochemically, it has been shown that classical neurotransmitters suchas noradrenaline (NA) (Aston-Jones et al., 1991a; Berridge andWaterhouse, 2003), acetycholine (Jones, 1991; Millan, 2003), serotonin(Millan, 2003; Ursin, 2002), glutamate (Chojnacka-Wojcik et al., 2001;Jones, 2003) and GABA (Gottesmann, 2002) are important transmitters ofarousal systems and also play important roles in regulating emotionalstates as they relate to anxiety-like behavior. In addition, variousneuropeptides such as hypocretin/orexin(Hcr/Ox) (Sutcliffe and de Lecea,2002), neuropeptide Y (Silva et al., 2002), galanin (Bing et al., 1993;Holmes et al., 2003; Saper et al., 2001), or nociceptin/orphanin FQ(Reinscheid and Civelli, 2002) are also modulators of arousal and/oranxiety. Anatomically, the dorsolateral pontine tegmental region is oneof the important areas that have been implicated in both sleepregulation and stress-related behaviors. The dorsolateral tegmentalregion contains several distinct nuclei such as Barrington's nucleus,the locus coeruleus (LC) and also comprises unidentified neurons outsideof the LC proper such as the peri-LC region (Rizvi et al., 1994; Sutinand Jacobowitz, 1988). The LC is the primary source of noradrenergicinput to the cortex and the NA-LC system plays important roles inregulating arousal and anxiety (Berridge and Waterhouse, 2003; Swansonand Hartman, 1975). Firing of LC neurons correlates with vigilancestates. Tonic discharge of LC neurons is virtually absent during rapideye movement (REM) sleep, low during slow wave sleep (SWS stages 1 and2) and highest during wakefulness (Foote et al., 1980; Hobson et al.,1975). Barrington's nucleus, the pontine micturition reflex center,expresses corticotrophin-releasing factor (CRF) as its peptidergicneurotransmitter (Sutin and Jacobowitz, 1988; Swanson et al., 1983;Valentino et al., 1995).

In addition to these known neurotransmitters and neurocircuities thatare involved in arousal and anxiety, there could be other importantregulators and structures in the CNS that have not yet been uncovered.Novel neurotransmitters or modulators can be found by using orphan Gprotein-coupled receptors (GPCRs) as targets. Orphan GPCRs are clonedreceptor proteins whose endogenous ligands have not yet been identified.Identification of the natural ligands (deorphanization) of orphan GPCRsleads to the discovery of novel neurotransmitters or modulators. Usingorphan GPCRs several novel neuropeptides have recently been discoveredwhich ultimately have shed new insights on our understanding ofparticular brain functions and helped to reveal novel therapeutictargets for mental disorders.

Described herein are certain physiological functions of such a newlydeorphanized GPCR system, NPS, and its cognate GPCR. The sequence of theGPCR (Genbank accession no. BD183774, BD183814, BD183773) was firstdisclosed in a patent published in April 2002 ((WO 02/31145 A1) (Sato,2002). The patent also reported the isolation of its endogenous peptideligand without providing further information about pharmacologicalcharacteristics and physiological functions. Here, we report that NPS isa novel neuropeptide that potently modulates arousal and could alsoregulate anxiety-related behavior. We further analyze the distributionof the NPS precursor mRNA expression and describe the existence of apreviously uncharacterized population of cells that are adjacent to thenoradrenergic LC neurons.

Evolutionary Conservation of NPS Primary Structures

The human, rat and mouse NPS precursor proteins contain a hydrophobicsignal peptide and a pair of basic amino acid residues preceding theunprocessed peptide. Searching public DNA databases we identified achicken EST clone and partial genomic sequences for the chimpanzee andcanine precursor proteins. Alignment of the deduced primary structuresof the mature peptide shows that the amino-terminal residue in allspecies is a conserved serine (FIG. 1). According to the nomenclaturethat has been used most recently (Shimomura et al., 2002), we propose toterm this novel peptide “Neuropeptide S” (NPS).

Pharmacological Profiles of NPS and NPSR

Cell lines stably expressing human NPSR in both Chinese hamster ovary(CHO) cells and human embryonic kidney 293 T cells (HEK 293T) were usedto define the pharmacological characteristics of NPS. Human, rat andmouse NPS induce dose dependent elevations in intracellular [Ca²⁺]_(i),in both HEK 293T (FIG. 2A) and CHO (data not shown) cell lines,indicating that the NPSR couples to G_(q) proteins. Half-maximaleffective concentrations (EC₅₀) for mobilization of [Ca²⁺]_(i), were9.4±3.2 nM, 3.2±1.1 nM and 3.0±1.3 nM for human, rat and mouse NPS,respectively.

Since position 10 of NPS is not conserved among the different species wedecided to substitute the corresponding amino acid by tyrosine (Y) inorder to develop an analog suitable for radioiodination. Human Y¹⁰-NPSretains full agonist activity with an EC₅₀ of 6.7±2.4 nM (data notshown). The monoiodinated form of Y¹⁰-NPS was used as a radioligand inreceptor binding experiments. Binding of [125I] Y¹⁰-hNPS to CHO cellsstably expressing hNPSR is saturable with high affinity (K_(d)=0.33±0.12nM; B_(max)=3.2±0.4 fmol/150.000 cells, FIG. 2B) and displaceable byincreasing concentrations of human NPS (IC₅₀=0.42±0.12 nM) (FIG. 2C). Nospecific binding was detected in mock-transfected CHO cells. Theseresults demonstrate that NPS binds and activates its cognate receptorwith high potency and specificity.

Distribution of NPS Precursor and Receptor mRNA Expression

We next examined the sites of synthesis of the NPS precursor andreceptor mRNA in rats. Quantitative RT-PCR shows that NPS and itsreceptor are expressed in various tissues, the highest levels beingfound in brain, thyroid, salivary and mammary glands (FIG. 3).

Since both NPS and NPSR mRNA are expressed highly in CNS among all thetissues examined, we next studied the localization of NPS and itsreceptor mRNA in rat brains by in situ hybridization. These experimentsrevealed that the rat NPS precursor mRNA is expressed discretely in afew brain areas, with strongest expression in the LC area (FIG. 4B),principle sensory 5 nucleus and lateral parabrachial nucleus (FIG. 5B,C). Moderate expression was also found in a few scattered cells of thedorsomedial hypothalamic nucleus (FIG. 5E, F) and the amygdala (FIG. 5H,I).

To describe the NPS expressing neurons in the LC area more precisely,double in situ hybridization with antisense probes for NPS precursor andtyrosine hydroxylase (TH) was carried out. As shown in FIG. 4C-E, NPSdoes not colocalize with TH. The majority of NPS positive cells wereobserved at midpontine levels, ventromedial to the noradrenergic LCneurons. Few NPS expressing neurons were found intermingled with THpositive cells at the ventral pole of LC proper. We conclude that theNPS expressing neurons in the LC area form a cluster of cells that donot produce NA and intermingle with LC proper neurons along the medialand ventral border of LC, extending just medially into the peri-LC area.

Within that area and ventromedial to the LC lies Barrington's nucleus,the micturition reflex center, which is a well-studied ovoid shapednucleus located at the rostral pole of LC. It has been shown thatBarrington's nucleus is negative for TH and choline acetyltransferaseand most of its neurons express CRF (Rizvi et al., 1994; Valentino etal., 2000). Double in situ hybridization with NPS and CRF antisenseriboprobes revealed that NPS does not colocalize with CRF (FIG. 4F-H).At the level of highest NPS neuron density, only a few scattered neuronswere found expressing CRF that were located ventrally to the NPSexpressing neurons. At a more rostral level, densely packed CRF positiveneurons were observed as the ovoid shaped Barrington's nucleus. Only fewNPS expressing neurons were found along the dorsal border ofBarrington's nucleus at this level. We conclude that the NPS expressingneurons lie caudally to Barrington's nucleus and at the mid-level of LC.They extend ventromedially from the LC proper, caudodorsally toBarrington's nucleus. This unique anatomical pattern of NPS expressingneurons defines a previously unrecognized population of cells located inbetween the noradrenergic LC proper and Barrington's nucleus.

The NPSR mRNA is widely expressed in many brain regions. The strongestexpression signals were found in several discrete nuclei or regions suchas anterior olfactory nucleus (FIG. 6B, C), dorsal and ventralendopiriform nucleus (FIG. 6B, C, E, H, I, K), amygdala (FIG. 6H, I),precommissural nucleus, paraventricular thalamic nucleus and subiculum(FIG. 6K, L). High levels of expression were also observed in corticalregions. Motor cortex 2 and retrosplenial agranular cortex are distinctareas in cortex that show strong expression of NPSR mRNA (FIG. 6E, H, K,M, O). Medium levels of expression are also found in dispersed neuronsin other cortical regions such as somatosensory cortex (FIG. 6N). Highlevel of expression was found in multiple nuclei of the hypothalamus(FIG. 6H, K). Moderate NPSR expression was also found in midbrain. Ponsand medulla are brain regions that express NPSR mRNA only weakly (datanot shown).

These data suggest that NPS could be involved in a variety of brainfunctions. Interestingly, NPSR mRNA is not detected in LC area. However,significant NPSR expression is also found in thalamic midline nucleisuch as central medial thalamic nucleus, interanteriomedial thalamicnucleus, reuniens and rhomboid thalamic nucleus (FIG. 6E), which relayextensive inputs from brain stem reticular formation to diffuse corticalfields and are involved in regulation of arousal and wakefulness (Vander Werf et al., 2002).

NPS Increases Locomotor Activity and Promotes Wakefulness

In view of the NPSR sites of expression and the prominent expression ofthe NPS precursor in LC area, we hypothesized that NPS may be involvedin arousal and anxiety. To start this investigation, we tested theeffects of NPS on locomotor activity in both naive and habituated mice(FIG. 7A). 0.1 nmole or 1 nmole NPS administeredintracerebroventricularly (i.c.v.) caused a significant increase inlocomotor activity in both naïve and habituated mice (p<0.01) during the60 min observation period, while 10 pmoles NPS did not. The totaldistance traveled, percentage of time moving, number of rearing eventsand center entries were also significantly increased in mice injectedwith 0.1 and 1 nmole NPS (data not shown). The elevation of locomotoractivity in habituated animals indicates that NPS may produce behavioralarousal independent of novelty or stress.

The effects of NPS on locomotor activity suggest a possible role of NPSin modulating sleep-wake patterns. Rats were implanted with a standardset of electrodes and electroencephalograms (EEG) and electromyograms(EMG) were recorded after i.c.v. administration of NPS. Polygraphicrecordings of vigilance states indicate that rats treated with 0.1 nmoleand 1.0 nmole of NPS spent up to 69% and 87%, respectively, of the firsthour of recording in wakefulness, compared to 45% for saline treatment(F_(2,21)=16.80; p<0.01) (FIG. 7B). In contrast, the amount of slow wavesleep stage 1 (SWS1) (F_(2,21)=9.69; p<0.01), stage 2 (SWS2)(F_(2,21)=11.859; p<0.01) and REM sleep (F_(2,13)=12.29; p<0.01) in NPStreated rats was significantly reduced compared with saline treatedanimals. The increase in wakefulness was due to a significant increasein the mean duration of the episodes (F_(2,21)=7.22; p<0.01), comparedto saline group. Interestingly, the increase in wakefulness during thefirst hour post NPS injection was followed by a rebound in the amount ofnon-REM sleep at the second hour (20% increase vs. saline(F_(2,21)=5.44; p<0.01)) and fourth hour (48% increase compared tosaline treated animals (F_(2, 21)=12.22; p<0.01)). Together, these datashow that NPS can promote arousal and might be involved in the inductionof wakefulness or suppression of sleep.

NPS Attenuates Anxiety-like Behavior

The expression of NPSR in several brain regions that are known to beinvolved in anxiety such as amygdala, thalamus and hypothalamic regionsindicates that the NPS system could also play a role in behavioralresponse to stress (Charney and Deutch, 1996; Redmond and Huang, 1979;Sah et al., 2003). Naïve mice were tested in the open field, a paradigmof free exploratory behavior in a novel environment. It was found thatNPS significantly increased the number of entries in the central zoneduring the first 10 minutes, which could indicate an anxiolytic-likeeffect (p<0.05; FIG. 8A). However, the same doses of NPS also increasedambulations in the outer zones of the open field, consistent with thearousal-promoting effect of the peptide. In order to further study NPSeffects on stress as it relates to anxiety, two additional tests wereperformed that are based on the natural aversion of rodents to open orunprotected spaces: the light-dark box and the elevated plus maze (FIG.8B, C). Mice injected with NPS exhibited a dose-dependent reduction inanxiety-like behavior in both paradigms.

In the light-dark box, mice injected with NPS at a dose range of 0.03- 3nmole, but not at 0.01 nmole, spent a prolonged time in the light area(p<0.05-0.01, FIG. 8B) and showed a higher percentage of entries in thelight area (data not shown). The latency until the first exit from theprotected dark compartment was significantly reduced by NPS at dosesbetween 0.3-3 nmole. General activity was also enhanced as the number oftransitions between the two compartments significantly increased atdoses between 0.1-3 nmole. In the elevated plus maze, mice injected with0.1 and 1 nmole NPS, but not at 0.01 nmole, spent significantly moretime on the open arms (p<0.05, FIG. 8C) and showed a higher number oftransitions from closed to the open arms (p <0.05-0.01). The averagenumber of transitions between the two closed arms of the elevated plusmaze (closed-closed transitions) was increased at all doses, but did notreach statistical significance. Closed-closed transitions are a measureof general activity in this behavioral paradigm, so our data indicatethat in the elevated plus maze NPS may not produce significanthyperlocomotion. Together, the increased number of entries and prolongedtime spent in the unprotected zones of both paradigms (open arm/lightarea) suggest that central administration of NPS produces ananxiolytic-like effect. However, consistent with the hyperlomocotoreffect of NPS as described above, these NPS doses (>0.1 nmole) alsosignificantly increased the total activity in both tests.

Many anxiolytic drugs increase exploratory activity in the open field,light-dark box or elevated plus maze paradigms. However, compoundsstimulating locomotion could produce false-positive effects in thesetests because the enhanced exploration could be secondary to theincrease in general activity. In order to validate the observedanxiolytic-like effects of NPS, we tested increasing doses of NPS in themarble-burying paradigm. Mice tend to bury objects such as glass marblespresent in their environment. Anxiolytic drugs such as benzodiazepinesreduce the number of marbles buried over a fixed period of time. It hasbeen suggested that the inhibition of marble-burying behavior iscorrelated with anxiolytic-like activity (Njung'e and Handley, 1991). Asshown in FIG. 8D, mice injected with saline covered about 50% of themarbles during the 30 min observation period (total of 18 marbles percage). NPS dose-dependently reduced the number of marbles buried.NPS-injected mice were actively exploring the marbles and eventuallyengaged in burying them, however at significantly lower numbers ascompared to mice injected with saline (p<0.05-0.01). In summary, thecombined results of all four paradigms measuring anxiety-like behaviorsuggest that NPS might produce anxiolytic-like effects in the presenceof increased arousal.

The effects of NPS on inducing wakefulness are rapid (during the firsthour after injection) and potent since low doses of NPS are sufficientto reduce all sleep stages such as REM, SWS1, and SWS2, suggesting aprofound change in sleep architecture. Recently, the neuropeptidehypocretin 1/orexin A (Hcrt/Ox) has also been demonstrated to inducearousal and genetic analysis has provided compelling evidence thatabsence of Hcrt/Ox or its receptor(s) produces narcolepsy in mice, dogsand humans (Sutcliffe and de Lecea, 2002). Single i.c.v. injection ofHcrt/Ox produces arousal lasting for 2-3 hours (Bourgin et al., 2000;Hagan et al., 1999) whereas comparable NPS administrations show a moreshort-term effect within the first hour post injection. Both peptidesappear to increase wakefulness while suppressing REM sleep and deepsleep (SWS stage 2) (Bourgin et al., 2000), although one study could notdetect a significant effect of Hcr/Ox on deep sleep duration (Hagan etal., 1999). Hcrt/Ox appears to exert its effects partially by directlyactivating noradrenergic LC neurons since orexin 1 receptors are foundto be colocalized with TH in LC neurons and electrophysiologicalrecordings from LC neurons show excitatory effects of exogenouslyapplied Hcrt/Ox (Bourgin et al., 2000). However, the arousal-promotingeffect of NPS is unlikely mediated by direct activation of noradrenergicsystems since our anatomical data show that NPS expressing neurons donot produce NA and no NPSR mRNA was detected in LC neurons. However, wecannot rule out an indirect activation of noradrenergic systems.Electrophysiological recording will be necessary to confirm a possiblelink between NPS and monoaminergic transmitter systems that have beenimplicated in the neurochemistry of wakefulness and arousal.

One unexpected outcome of this study is the discovery of a cluster ofNPS expressing neurons that do not produce NA or CRF and are localizedin close proximity to the LC proper and Barrington's nucleus. Thecluster of NPS expressing neurons is likely to be a previouslyuncharacterized population of cells in the peri-LC area. Noteworthy, ithas been reported before that a large number of uncharacterized neuronsare found in the peri-LC area (Aston-Jones et al., 1991b; Rizvi et al.,1994) and our present data suggest that the NPS neuronal cluster couldbe a subset of these neurons.

It is well documented that the noradrenergic LC is involved in theregulation of an aroused state of wakefulness (Berridge and Waterhouse,2003). On the other hand, several studies found no major disruption ofEEG activity after selective cytotoxic lesions of TH-positive LC neuronsor genetic ablation of the noradrenaline-synthesizing enzyme dopaminebeta-hydroxylase (Cirelli et al., 1996; Hunsley and Palmiter, 2003),underscoring the fact that arousal is modulated by multiple neuronalsystems. Our present data provide evidence that NPS could be a novelarousal-modulating transmitter system. Interestingly, the close vicinityof NPS producing neurons and the noradrenergic neuronal cluster in LCindicate that this brainstem area might contain two independenttransmitter systems that regulate vigilance states.

Central administration of NPS produces anxiolytic-like effects but alsoincreases locomotor activity at similar doses. In the open filed,elevated plus maze and light dark box paradigms, increases inexploration are generally interpreted as an anxiolytic effect but theinterpretation might be confounded by hyperlocomotion. Factor analysis,however, has shown that the behavioral parameters monitored in thesetests can be divided into two components: an activity component (totaldistance traveled, number of transitions) and an anxiety component(number of entries in unprotected zone, time spent in unprotected zone)(Rodgers and Johnson, 1995) and that these two components show poorcorrelation. For example, both the psychostimulants amphetamine andcocaine produce hyperlocomotion yet increase anxiety-like behavior, i.e.they are anxiogenic (Hascoet and Bourin, 1998; Paine et al., 2002). Onthe other hand, the wake-promoting neuropeptide Hcrt/Ox enhances arousaland hyperlocomotion and suppresses REM sleep , but has no effect onanxiety-like behavior in rodents (Hagan et al., 1999). Moreover, typicalanxiolytic drugs, such as benzodiazepines, have either no effect orreduce locomotor activity, depending on the doses used (Chaouloff etal., 1997). Therefore, although we acknowledge the possible confoundingeffects of hyperlocomotion, we suggest that the increased exploratoryactivity observed in mice after NPS administration may indicate ananxiolytic-like profile in these three paradigms. To further investigatethe possible anxiolytic-like effects of NPS, we used the marble-buryingtest as an alternative behavioral paradigm. In this test, the selectivesuppression of marble burying behavior is suggested to correlate withanxiolytic activity, in contrast to the other three paradigms whereincreases of natural behaviors are an index of anxiolytic-like effects.Numerous drugs clinically effective in the treatment of anxietydisorders such as benzodiazepines or selective serotonin reuptakeinhibitors reduce marble burying behavior in rodents (Borsini et al.,2002). Our data demonstrate that NPS also inhibits this natural behaviorat doses which increase locomotion. Altogether, central administrationof NPS reduces behavioral signs of anxiety in four different anxietytests. These findings indicate that NPS could be involved in modulatinganxiety responses.

Thus, central administration of NPS produces a unique behavioral profileby increasing locomotor activity and wakefulness in rodents. NPS couldalso exert anxiolytic-like effects. In addition, we identify apreviously undescribed group of neurons adjacent to the noradrenergic LCthat express NPS. The discovery of this novel transmitter system thatmodulates sleep-wake cycles and anxiety might help to further ourunderstanding of sleep disorders, such as insomnia, and pathologicalstates of anxiety. It should be noted that excessive anxiety anddisruption of sleep patterns are often observed in patients sufferingfrom depression and, thus, the methods of the present invention may alsobe useable in the treatment of depression and/or depression relatedanxiety or sleep disturbances.

Molecular Cloning of Human NPSR and Rat NPS Precursor

Human NPSR was cloned into pcDNA3.1(+) from human brain cDNA (Clontech,Carlsbad, Calif.) using nested PCR. Primers were5′-aggagcaaggacagtgaggctcaa-3′ (SEQ ID NO: 11) and5′-tgcccaagcaggtgacaaggacct-3′ (SEQ ID NO: 12) for first roundamplification and 5′-atactcgagccatgccagccaacttcacagagggca-3′ (SEQ ID NO:13) and 5′-gcttctagagctcagcctagcactggcactgcccta-3′ (SEQ ID NO: 14) forsecond round. Rat NPS precursor cDNA was cloned into pBluescript from arat total brain cDNA library (Clontech). First round amplificationprimers were 5′-cagattttgggaagtcca-3′ (SEQ ID NO: 15) and5′-agattaattccccgagtc-3′; (SEQ ID NO: 16) second round primers were5′-gtttctagaaatgattagctcagtaaaactcaa-3′ (SEQ ID NO: 17) and5′-gcagaattcgtcatgattttgctctttgaaagg-3′ (SEQ ID NO: 18). The cloned DNAswere sequenced on both strands.

Cell Transfection and Intracellular Ca²⁺ Measurement

HEK 293T cells and CHO dhfr(−) cells were transfected with the humanNPSR cDNA cloned into pcDNA 3.1(+) using LipofectAmine. Stable cloneswere selected with 800 mg/l G418 and tested for mobilization ofintracellular Ca²⁺ with 100 nM NPS (generous gift of PhoenixPharmaceuticals, Belmont, Calif.). Changes in intracellular Ca²⁺ weremeasured in a fluorometric imaging plate reader system (FLIPR, MolecularDevices) as described before(Saito et al., 1999). Dose response curvesfor agonist activation were calculated from peak fluorescence values oftriplicate incubations and EC₅₀ values were calculated with Prismsoftware (GraphPad, San Diego, Calif.).

Radioligand-binding Assay

Y¹⁰-NPS was labeled with ¹²⁵I using the chloramine T method and purifiedby reversed-phase HPLC in a collaboration with NEN Perkin Elmer (Boston,Mass.). CHO cells stably expressing human NPSR were seeded into 24-wellplates and cultured for 48 hours. For saturation binding experiment,[¹²⁵I] Y¹⁰-NPS at concentrations from 4 pM to 1.7 nM were used. Fordisplacement binding, increasing concentrations of unlabelled human NPS(1 pM to 3 μM) were used to compete with 0.15 nM [¹²⁵I] Y¹⁰-NPS.Nonspecific binding was determined in the presence of 1 μM unlabeledhuman NPS. The binding assay was carried out as described (Sakurai etal., 1998). In brief, cells were washed with PBS first and thenincubated with radioligand with or without unlabeled NPS peptide in DMEMmedium containing 0.1% bovine serum albumin at 20° C. for 1.5 h. Cellswere washed 5 times with cold PBS and lysed with 1 N NaOH. Boundradioactivity was counted in a MicroBeta liquid scintillation counter(EG&G Wallac, Gaithersburg, Md.) and corrected for counting efficiency.Data from triplicate incubations were analyzed using PRISM.

Quantitative Real-time PCR

Tissue was collected from male and female adult Sprague Dawley rats andRNA was extracted with Trizol. PolyA+RNA was prepared using OligoTex(Qiagen) and converted to cDNA using oligo dT and random primers withSuperscript reverse transcriptase (Invitrogen). Primers for NPSR(5′-tgcagggagcaaagatcaca-3′ (SEQ ID NO: 19) and5′-aatctgcatctcatgcctctca-3′(SEQ ID NO: 20)) , NPS precursor(5′-tgtcgctgtccacaatgcat-3′ (SEQ ID NO: 21) and5′-aatcagattttccagacaccttagaag-3′ (SEQ ID NO: 22)) and β-actin(5′-cacggcatcgtcaccaact-3′ (SEQ ID NO: 23) and 5′-agccacacgcagctcattg-3′(SEQ ID NO: 24)) were predicted using ABI Prism Primer Select softwareand tested for linearity of amplification using cloned cDNAs astemplate. Quantitative real-time PCR was performed in an ABI Prism 7000using SYBR Green PCR Master Mix (Applied Biosystems).

In Situ Hybridization

A 326 bp fragment of the rat NPSR (corresponding to nt 408-734) wasamplified by PCR and subcloned into pBluescript SK. A fragment of therat NPS precursor (corresponding to nt 92-276) was cloned into the samevector. Sense and antisense riboprobes were labeled with ³⁵S-UTP. Rattyrosine hydroxylase (TH) cDNA was a gift from Dr. Francis Leslie (UCI)and cloned in pBluescript. Rat corticotropin-releasing factor (CRF) cDNAwas a gift from Dr. Christine Gall (UCI) and cloned in the same vector.For double in situ hybridization, antisense TH riboprobes or CRFriboprobes were labeled with digoxigenin using DIG RNA labeling kit(Roche Applied Science). In situ and double in situ hybridization to 20μm coronal sections of adult Sprague Dawley rat brains was carried outas described before (Clark et al., 2001).

Behavioral Studies.

Male C57Bl/6 mice (National Cancer Institute, Bethesda, MD), age 10-14weeks, were group-housed (4 animals per cage) under controlledconditions (temperature 21±2° C.; relative humidity 50-60%; 12-hourlight-dark cycle, lights on 6:00 AM) with free access to food and water.Prior to drug injections, mice were briefly anesthetized with halothane.NPS was dissolved in phosphate-buffered saline (PBS, pH 7.4) andinjected i.c.v. (total volume: 2 μl) as described before (Laursen andBelknap, 1986). Mice were allowed to recover for 5 min and then placedin the observation apparatus.

For sleep studies, adult male Sprague-Dawley rats (250-300 g) wereimplanted under halothane anesthesia (1-2%) with a stainless steelcannula for i.c.v. administration and a standard set of stainless steelscrew electrodes for chronic sleep recordings as reported previously(Bourgin et al., 2000). Rats were injected with NPS or vehicle (5 μl) atthe beginning of the light cycle and cortical activity was recorded over6 hours. All animal experiments had been approved by the local IACUCcommittee and were done in accordance with federal regulations andguidelines on animal experimentation.

Locomotion was monitored in rectangular plexiglass boxes (60×40×50 cm).Horizontal activity was measured over 60 min by 18×12 infrared sensorsplaced 2 cm above the floor. A second row of sensors at 8 cm above thefloor was used to record rearing events. The imaginary central zone wasdefined as a 30×20 cm rectangle in the middle of each observation area.Data were collected using MatLab (Mathworks, Natick, Mass.).Experimental procedures for open field, elevated plus-maze andlight-dark box were described previously (Köster et al., 1999). Marbleburying was measured in mice placed individually in polypropylene cages(30×18×12 cm) containing 18 glass marbles (1.5 cm diameter) evenlyspaced on 5 cm deep rodent bedding (bed-o'cob, The Andersons Inc.,Maumee, Ohio) (Njung'e and Handley, 1991). No food or water was presentduring the observation period. Cages were covered with a metal grid andthe number of marbles covered at least two-thirds was counted after 30min.

Applicants have determined that a number of single-nucleotidepolymorphisms in the NPSR gene are associated with increased risk ofasthma and possibly other forms of atopic diseases but the physiologicalconsequences of the mutations remain unclear. As explained in thefollowing paragraphs, one of the polymorphisms produces an Asn-Ile¹⁰⁷exchange in the second extracellular loop of the receptor protein and aC-terminal splice variant of the NPSR was found over-expressed in humanasthmatic airway tissue. Described herebelow are studies wherein the tworeceptor variants of the NPSR were compared with the wildtype protein.The Asn-Ile¹⁰⁷polymorphism is determined to result in a gain-of-functioncharacterized by an increase in agonist potency without changing bindingaffinity. In contrast, the C-terminal splice variant of the NPSR shows apharmacological profile similar to the wildtype receptor. The alteredpharmacology of the Ile¹⁰⁷ isoform of the NPSR implies a physiologicalmechanism of enhanced NPS signaling that could contribute to thepathophysiological changes observed in asthmatic airway tissue.

Asthma is characterized by increased constriction of smooth muscles inbronchial airways, airway inflammation accompanied by activation ofmacrophages and mast cells followed by mucus hypersecretion and finallyairway remodeling. Therapeutically, reduction of smooth muscle tone byadrenergic agonists and suppression of inflammatory processes byglucocorticoids and cysteinyl-leukotriene receptor (CysLT) antagonistsremain the preferred treatment options, although both strategies areonly symptomatic. A number of mechanisms have been discussed regardingthe pathophysiology of asthma. Large epidemiological studies havepointed at both genetic and environmental factors to increase the riskof developing asthma and recent efforts in positional cloning haveidentified a number of candidate genes that might representsusceptibility factors. However, in most cases the physiologicalfunction of these candidate genes and their mechanism of action remainelusive.

Very recently, a G protein-coupled receptor was identified in Finnishand Canadian asthma patients that was linked to an increasedsusceptibility for asthma and potentially other forms of allergy thatare characterized by high IgE serum levels. The study described a numberof risk haplotypes and originally termed the receptor “GPRA isoform A”(for G protein-coupled receptor associated with asthma, GenBankaccession no. NP_(—)997055; the protein has also been termed GPR 154).This receptor protein is identical to the NPSR that we have studiedextensively for its pharmacology, distribution and function in brain. Asingle-nucleotide polymorphism associated with the increased riskhaplotype changes the primary structure of the receptor protein to codefor an Asn-Ile exchange at position 107 of the mature protein (SNP591694A>T; refSNP ID: rs324981). The study also found that a C-terminal splicevariant of the receptor, originally termed “GPRA isoform B” (GenBankaccession no. NP_(—)997056), was over-expressed in human asthmaticairway tissue and that the orthologous murine receptor mRNA wasupregulated in a mouse model of chronic airway inflammation. Mostimportantly, bronchial smooth muscle cells were shown to express thereceptor, indicating a potential role in bronchial constriction. De novoexpression of the C-terminal splice variant was detected in asthmaticbronchial biopsies while expression levels of the wildtype receptorprotein (synonymous: GPRA isoform A) were significantly lower in healthyairway smooth muscle. These data strongly suggest a possible involvementof the mutant receptor in the pathophysiology of asthma, but the reportby Laitinen et al. did not describe a functional role for the receptoror its isoforms due to the lack of a pharmacologically useable agonist.

Naturally occurring NPS is a peptide whose conserved structure carriesan aminoterminal serine residue in all species thus far examined. Theprimary structure of the wildtype NPSR (NPSR WT) is identical to isoformA of GPRA. The receptor variant containing isoleucine at position 107 isreferred to in this patent application as “NPSR Ile¹⁰⁷” (NPSR Ile¹⁰⁷)and the splice variant containing an alternative C-terminal exon istermed “NPSR C-alt.”

In initial studies Applicants found that NPSR is widely expressed in therat brain while the NPS precursor mRNA is found in only a few brainstructures. Highest levels of NPS precursor expression were detected ina novel nucleus located in between the noradrenergic locus coeruleus andBarrington's nucleus in the pontine area of the rat brain stem. Otherbrain regions of high NPS precursor expression include the lateralparabrachial nucleus, sensory principle 5 nucleus and a few scatteredneurons in the amygdala and dorsomedial hypothalamic nucleus. Inaddition, we found high expression of NPS and NPSR mRNA in endocrinetissues including thyroid, mammary and salivary glands but did notobserve significant levels in rat lung tissue.

Central administration of NPS promotes behavioral arousal and suppressesall stages of sleep in rodents. Furthermore, NPS was found to produceanxiolytic-like effects in a battery of four different tests thatmeasure behavioral responses of rodents to stress. NPS was shown toinduce transient increases of intracellular Ca²⁺, indicating that itmight have excitatory effects at the cellular level.

Even though a number of polymorphisms were described for NPSR andalthough each of them could have functional significance, an immediatestructural change of the receptor protein is produced by the pointmutation at amino acid position 107 (Asn-Ile¹⁰⁷) and the use of analternative 3′ exon. Therefore, the present study focused oninvestigating if the point mutation or alternative splicing of NPSRcould affect the pharmacological profile of the receptor and if thesechanges might have functional consequences that could indicate apossible physiological role of NPSR variants in bronchial tissue. Inthis study we demonstrate that a gain-of-function mutation in NPSRIle¹⁰⁷ could explain some of the pathological mechanisms of asthma.

Cloning and Functional Expression of NPSR Isoforms

Human NPSR WT cDNA was cloned using known techniques, as described inXu, Y.-L., Reinscheid, R. K., Huitron-Resendiz, S., Clark, S. D., Wang,Z., Lin, S. H., Brucher, F. A., Zeng, J., Ly, N. K., Henriksen, S. J.,de Lecea, L., and Civelli, O. (2004) Neuron Volume 43, Pages 487-497.The Ile¹⁰⁷ isoform of NPSR was generated by PCR using syntheticoligonucleotides and the Quik Change Site-Directed Mutagenesis kit fromStratagene. The C-terminal splice variant of NPSR (NPSR C-alt) wasgenerated by recombinant PCR. First, the alternatively spliced exon wascloned by PCR from human genomic DNA using primers NPSRB5(5′-ATCTCTTTCCCCTGCAGGGTCATCCGTCTCC) (SEQ ID NO: 25) and NPSRB3-XbaI(5′-TTTCTAGAGAGCTGTCACCTTGGAA, XbaI site underlined) (SEQ ID NO: 26).Recombinant PCR was carried out with the cloned exon and human NPSR WTas templates using primers NPSRA5-XhoI (5′-ATACTCGAGCCATGCCAGCCAACTTCACAGAGGGCA, XhoI site underlined) (SEQ ID NO:27) and NPSRB3-XbaI. The products were gel-purified and cloned intopcDNA3.1 hygro (Invitrogen). Transfection of CHO and HEK 293 cells wascarried out using lipofectamine (Life Technologies) as described in Xu,Y.-L., Reinscheid, R. K., Huitron-Resendiz, S., Clark, S. D., Wang, Z.,Lin, S. H., Brucher, F. A., Zeng, J., Ly, N. K., Henriksen, S. J., deLecea, L., and Civelli, O. (2004) Neuron Volume 43, Pages 487-497.Selection of stable clones was achieved by culturing transfected cellsin medium containing 400 mg/l hygromycin (Omega Scientific, Tarzana,Calif.). For transient expression, transfected cells were cultured for72 hours without antibiotic selection.

Detection of Endogenous NPSR Expression in Cell Lines

Total RNA from a number of human cell lines (HEK293, U373, 1321N,Caco-2, LoVo, DLD-1, Colo205, HT-29, SW480, SW1116, HCT116) wasextracted and converted into single-strand cDNA using reversetranscriptase (New England Biolabs) and oligo dT primers. Quantitativereal-time PCR was carried out as described using primers specific forhuman NPSR.

Measurement of Intracellular Ca²⁺ Mobilization

Changes of agonist-induced intracellular Ca²⁺ were measured using theFLIPR technology as described before (5, 6). NPS and truncated NPSpeptides were a gift from Phoenix Pharmaceuticals (Belmont, Calif.).Dose-response curves were calculated using GraphPad Prism (Graph Pad,San Diego). Mean EC₅₀ values of populations of stable clones expressingeither wildtype or mutant NPSR were compared using unpaired t-test andp<0.05 was considered significant.

Measurement of cAMP Accumulation

Accumulation of cAMP was measured in accordance with known technique.Stably transfected cells were seeded into 24-well plates and culturedfor 24 hours. Culture medium was aspirated and exchanged for 200 μlOpti-MEM (Life Technologies) containing 100 μM3-isobutyl-1-methylxanthine and agonists at various concentrations.After incubation for 15 min at 37° C. cells were lysed by adding ethanolto a final concentration of 66%. Aliquots of the lysate were lyophilizedand cAMP content was measured in a radioimmunoassay (either Flashplate,NEN Perkin Elmer, or SPA Biotrak, Amersham). Dose response curves werecalculated using GraphPad Prism.

CRE-Luciferase Reporter Gene Assays

HEK 293 cells were stably transfected with a reporter gene containing 6copies of a cAMP response element (CRE; sequence CCAAT) in front of aluciferase reporter gene (Promega) cloned into pcDNA3.1 neo. One stableclone showing robust induction of reporter gene expression afterforskolin challenge was chosen for further experiments. The differentNPSR isoform cDNAs (cloned into pcDNA3.1 hygro) were transfected intothese cells using lipofectamine and stable clones were selected. Thesame cells and procedures were also used for transient transfection. Forinduction of reporter gene expression cells were plated in 96-wellplates and incubated with agonist for 5 hours in serum-free medium,followed by aspiration of the medium and cell lysis with 25 mMTris-phosphate, pH 7.8, 2 mM dithiothreitol, 2 mM1,2-diaminocyclohexane-N,N,N′,N′,-tetraacetic acid, 10% glycerol, 1%Triton X-100. After one freeze-thaw cycle aliquots of supernatant weretransferred to white clear-bottom 96-well plates (Greiner) andluciferase content was quantified by bioluminescence (Luc-Screen,Applied Biosystems). Plates were counted in a scintillation counterusing bioluminescence settings (MicroBeta, Wallac-Perkin Elmer).

Cell Proliferation Assays

Cells were seeded in 24-well plates and grown overnight to 60-70%confluency. Following 24 h serum-starvation, 2 μCi of methyl-[³H]thymidine was added together with increasing concentrations of NPS. 500nM PGE₂ served as a positive control. After 3 h incubation cells werewashed 3 times with PBS and then lysed with 0.5 N NaOH. The lysate wasneutralized with 0.5 N HCl and aliquots were counted in a liquidscintillation counter for incorporated radioactivity.

MAP Kinase Phosphorylation

Agonist-induced phosphorylation of p42/p44 mitogen-activated proteinkinase (MAPK) was determined as described in Saito, Y., Wang, Z.,Hagino-Yamagishi, K., Civelli, O., Kawashima, S., and Maruyama, K.(2001) Biochem. Biophys. Res. Commun. Volume 289, Pages 44-50. Briefly,cells were cultured in 24-well plates in serum-free cell culture mediumfor 24 h. After stimulation for 5 min at 37° C. with increasingconcentrations of NPS, cells were washed with phosphate-buffered salineand lysed with 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.1% SDS,1.5% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM sodium orthovanadate,1 mM sodium fluoride, 0.5 mM phenylmethylsulfonyl fluoride, 1 μg/mlaprotinin, 0.5 μg/ml leupeptin, 0.7 μg/ml pepstatin, 100 g/mlbacitracin. Lysates were centrifuged and aliquots of supernatantelectrophoresed on 4-10% SDS-polyacrylamide gels. Phosphorylated p42/p44MAPK was assayed by Western Blot. After transfer to Hybond C membranes(Amersham), blots were incubated with anti-phospho p42/p44 MAPK antibody(Cell Signaling Technologies; dilution 1:1500) in Tris-buffered saline,1% nonfat dried milk, 0.2% Tween 20 at 4° C. overnight. Horseradishperoxidase-conjugated goat anti-rabbit IgG (Jackson Immuno Research Lab;dilution 1:2000) was used as a secondary antibody. Immunoblots weredeveloped using an Enhanced Chemiluminescence detection kit (Amersham)and films were scanned by densitometry (UN-SCAN-IT; Silk ScientificInc., Orem, Utah) for quantitative analysis.

Radioligand Binding

Saturation binding experiments in intact cells were carried out asdescribed in Xu, Y.-L., Reinscheid, R. K., Huitron-Resendiz, S., Clark,S. D., Wang, Z., Lin, S. H., Brucher, F. A., Zeng, J., Ly, N. K.,Henriksen, S. J., de Lecea, L., and Civelli, O. (2004) Neuron, Volume43, Pages 487-497. K_(d) and B_(max) of [¹²⁵I] Y¹⁰-NPS binding werecalculated using GraphPad Prism. [¹²⁵I] Y¹⁰-NPS was a gift from NENPerkin-Elmer. Non-specific binding was determined in the presence of 1μM NPS.

NPS produces an increase in intracellular Ca²⁺ in cells stablyexpressing NPSR WT with an EC₅₀ around 5-10 nM. Cells transientlytransfected with either wildtype, NPSR Ile or NPSR C-alt cDNAs did notshow agonist-induced changes in Ca²⁺ (data not shown). Also, we couldnot detect significant receptor binding in transiently transfected cellswith any of the constructs, indicating that the proteins might be eitherdifficult to express or expressed at low levels. Therefore, weestablished stable cells lines expressing either NPSR WT or NPSR lie inHEK 293 cells.

Two cells lines expressing similar levels (measured by radioligandbinding; see below) of either NPSR WT or NPSR Ile were chosen fordetailed analyses of pharmacological parameters. As shown in FIG. 10A,NPS induced a dose-dependent increase in intracellular free Ca²⁺ in bothcell lines. Cells expressing NPSR WT displayed an EC₅₀ of 12.4±1.24 nM,whereas the clone expressing NPSR lie responded to agonist stimulationwith an EC₅₀ of 1.4±1.17 nM. In order to investigate whether thistenfold increase in agonist potency at NPSR lie was a general propertyof the receptor protein we compared a large number of stable clonesexpressing either NPSR WT or NPSR Ile¹⁰⁷ by establishing dose-responsecurves for each construct and calculating mean EC₅₀ values for thepopulations. As shown in FIG. 10B, the mean EC₅₀ for NPS-inducedmobilization of intracellular Ca²⁺ at NPSR WT was found to be 13.02±1.18nM (n=16 individual stable clones). In contrast, NPSR Ile¹⁰⁷ displayed amore than ten-fold lower mean EC₅₀ of 1.01±0.13 nM (n=21 individualstable clones). These data demonstrate that NPSR Ile¹⁰⁷ is activated bysignificantly lower concentrations of agonist (F=3.365, p=0.0101).

Using the same approach as described before, Applicants compared theEC₅₀ values of a large number of stable clones established in HEK cellsthat co-express the CRE-Luciferase reporter gene. As shown in FIG. 10B,HEK cells stably expressing both the wildtype NPSR and the reporter genedisplayed a mean EC₅₀ of 45.08±1.19 nM for NPS-induced reporter geneexpression (n=7 individual clones). In contrast, cells co-expressingNPSR Ile¹⁰⁷ and the reporter gene showed a mean EC₅₀ of 0.63±0.43 nM forNPS (n=21 individual stable clones). Again, these data indicate thatNPSR Ile¹⁰⁷ is activated at significantly lower agonist concentrationsthan the wildtype receptor (F=5.854, p=0.0183). Since expression of thereporter gene is proportional to the amount of activated CREB proteinbinding to the CRE sequences within the reporter gene promotor, thesedata also suggest that NPSR is able to signal via G_(s)-type of Gproteins to increase cAMP formation. We confirmed this hypothesis bydirectly quantifying cAMP levels in the two stable clones that were usedpreviously to establish dose-response relationships for mobilization ofintracellular Ca²⁺. As shown in FIG. 11A, NPS induced cAMP accumulationin HEK cells stably expressing the wildtype receptor with an EC₅₀ of31.9±1.17 nM. In HEK cells expressing NPSR Ile¹⁰⁷, NPS stimulated cAMPformation at about ten-fold lower agonist concentrations with an EC₅₀ of3.45±1.26 nM.

As mentioned above, it was not possible to observe second messengersignaling in transiently transfected cells measuring either mobilizationof Ca²⁺ or formation of cAMP. However, due to the high rate of signalamplification of the luciferase reporter gene assay, it was possible toestablish agonist dose-response curves with this assay in transientlytransfected cells. As shown in FIG. 11B, the wildtype NPSR displayedonly a weak induction of luciferase activity with an EC₅₀ of 76.6±21.5nM. In contrast, NPSR Ile produced a robust increase in reporter geneexpression with an EC₅₀ of 17.3±1.31 nM. The NPSR variant containing thealternatively spliced C-terminus (NPSR C-alt) displayed an EC₅₀ of146.5±15.1 nM, very similar to the wildtype NPSR. The maximum ofreporter gene expression in HEK cells transiently transfected with NPSRIle¹⁰⁷ was about 2 fold higher than in cells expressing NPSR WT or NPSRC-alt, suggesting an increase in agonist efficacy as well as totalreceptor number. The magnitude of reporter gene induction was about20-fold lower in transiently transfected cells as compared to stableclones. Taken together, these data indicate that the Ile mutationproduces a gain-of-function in the NPSR protein, whereas thealternatively spliced C-terminus does not appear to affect secondmessenger coupling of the receptor.

Chronic asthma is accompanied by airway tissue remodeling, involvingproliferation of smooth muscle cell layers and basement membranehyperplasia. In order to investigate whether NPSR can affect cellproliferation at natural levels of receptor expression, we screened anumber of cancer cell lines for endogenous NPSR mRNA expression byRT-PCR. The human colon cancer line Colo205 was found to express NPSRtranscripts but did not display agonist-induced second messengerresponses (data not shown). However, NPS produced a dose-dependentstimulation of thymidine incorporation in Colo205 cells, indicating thatthe peptide can stimulate cell proliferation and mitogenic signals inthese cells (FIG. 12A). Doses of 0.1-10 nM NPS produced maximalthymidine incorporation, exceeding the effect of the well-characterizedmitogen prostaglandin E2 on these cells. The human colon cancer cellline DLD-1, which does not express NPSR transcripts, served as anegative control and showed no NPS-dependent thymidine incorporation(data not shown). We next examined potential intracellular mediators ofthe proliferative response elicited by NPS. The p42/p44mitogen-activated protein kinase (MAPK) is a well-known integrator ofmitogenic signals and many GPCRs have been shown to increasephosphorylation of MAPK upon agonist stimulation. We found that NPS canstimulate MAPK phosphorylation in a dose-dependent manner in HEK cellsstably expressing NPSR WT or NPSR Ile¹⁰⁷ isoforms. As observed before,NPS was more potent to induce MAPK phosphorylation in cells expressingNPSR Ile¹⁰⁷ (EC₅₀: 0.32±0.25 nM) than in cells expressing NPSR WT (EC₅₀:1.23±0.38 nM) (FIG. 12B).

Two individual clones, expressing either NPSR WT or NPSR Ile¹⁰⁷ withEC₅₀ values close to the average EC₅₀ in mobilizing intracellular Ca²⁺,were chosen to examine the structure-activity relationships of variousNPS fragments. These NPS fragments represent potential processingproducts that could result from proteolytic cleavage or human NPS 1-20.In addition, we also tested the effect of rat and mouse NPS 1-20 onthese cells. As shown in FIGS. 9 and 13, most carboxy-terminallytruncated fragments of NPS retained full agonist potency at bothwildtype and NPSR Ile¹⁰⁷. Rat and mouse NPS 1-20 appear to be slightlymore potent agonists at both NPSR isoforms as compared to the humanpeptide. Interestingly, NPS 1-12 almost completely lost agonist activityat the wildtype receptor while still behaving as a full, but weaklypotent, agonist at NPSR Ile¹⁰⁷. Further deletion of the two lysineresidues (at position 11 and 12) produced NPS 1-10. NPS 1-10 displayedfull agonist activity at both NPSR isoforms, albeit with significantlyhigher potency at NPSR Ile¹⁰⁷. Deletion of the three amino-terminalamino acids (NPS 4-20) completely abolished agonist activity. These dataindicate that the N-terminus of NPS contains the pharmacophore. The twolysine residues at position 11 and 12 appear to prohibit activation ofthe receptor when exposed at the C-terminus because further C-terminaldeletion to NPS 1-10 restored agonist activity. Because of the peculiarpharmacology of NPS 1-12, this fragment was also tested for possibleantagonist activity but failed to block activation of the two receptorisoforms by NPS 1-20 (data not shown). Overall, the various NPSfragments display a 5-10 fold higher potency at NPSR Ile¹⁰⁷ as comparedto NPSR WT.

Some of the previous observations could be explained by an increasedaffinity or receptor expression of NPSR Ile¹⁰⁷ versus NPSR WT. In viewof this, Applicants determined receptor binding parameters in a numberof stable clones for both variants. Surprisingly, both NPSR WT and NPSRIle¹⁰⁷ bind the radioligand with very similar affinities (K_(d) range ofNPSR wildtype clones: 0.2-0.45 nM, n=4, average K_(d): 347.1±44 pM;K_(d) range of NPSR Ile¹⁰⁷ clones: 0.17-0.4 nM, n=4, average K_(d):402.5±49 pM). However, stable NPSR Ile¹⁰⁷ clones tended to express morefunctional receptor protein per cell than NPSR WT clones (averageB_(max) of NPSR Ile¹⁰⁷ clones: 12.5±3.5 fmole/10⁵ cells; average B_(max)of NPSR WT clones: 3.9±1.5 fmoles/10⁵ cells; n=4 for each receptorvariant). It is currently not known how the Asn¹⁰⁷Ile exchange caninfluence protein levels in transfected cells. In general, the levels ofNPSR expression are low compared to other GPCRs expressed in the samecellular environment. Dose-response relationship experiments for Ca²⁺mobilization, cAMP formation and MAPK phosphorylation were performedwith two individual clones that displayed very similar receptor levelsin order to correct for possible confounds caused by varying numbers offunctional receptors (FIGS. 10A, 11A, and 12B). Taken together, our dataindicate that the increased potency of NPS at NPSR Ile¹⁰⁷ is not causedby a change in receptor affinity but might reflect an increasedintrinsic efficacy of the receptor protein to couple to G proteins andthus to the various second messenger pathways, as described above. Inthe present study Applicants have investigated the generalpharmacological properties of three natural variants of the human NPSRthat has been recently identified as an asthma susceptibility gene.Applicants also sought to determine whether the coding polymorphism oralternative splicing of NPSR would affect the receptor pharmacology in away that might have functional significance for the pathophysiology ofasthma.

Applicants' data provide evidence for an increased agonist efficacy atNPSR Ile¹⁰⁷. Surprisingly, the Asn¹⁰⁷Ile point mutation does not affectligand binding affinity, even though this amino acid is expected to beclose to the ligand binding pocket of the receptor protein. Theendogenous agonist NPS displays about a ten-fold higher efficacy at NPSRIle¹⁰⁷ as compared to the wildtype receptor in mobilizing intracellularCa²⁺, stimulating cAMP formation or inducing MAPK phosphorylation. TheIle¹⁰⁷ mutation does not produce increased constitutive activity, asjudged from the cAMP accumulation and reporter gene assays. A plausibleexplanation for our observations could be that the Asn¹⁰⁷Ilepolymorphism is producing a conformational change of the receptorprotein that facilitates G protein interaction and thus increasesagonist efficacy. Also, we observed a trend to higher levels of receptorprotein expression in stable clones expressing NPSR Ile¹⁰⁷. However, itis not known if NPSR Ile¹⁰⁷ expression is also facilitated in vivo orour observation is due to the over-expression system used in ourstudies. Together, our data indicate that the Asn¹⁰⁷Ile polymorphismproduces a gain-of-function that could have significant functionalconsequences with regard to the pathophysiology of asthma.

The receptor protein appears to be expressed in airway smooth musclecells that might contribute to bronchial constriction. Applicant's dataindicate that activation of NPSR produces an increase in intracellularfree Ca²⁺ and that the NPSR Ile¹⁰⁷ variant, when expressed in airwaysmooth muscle cells, could thus transmit an enhanced contractileresponse requiring lower agonist concentrations. Since increasedbronchial constriction is one of the physiological hallmarks of asthma,the gain-of-function mutation in NPSR Ile¹⁰⁷ could therefore beassociated with this phenotype. Also, our studies provide evidence for aproliferative effect of NPS using a cellular model of endogenous NPSRexpression and demonstrating enhanced phosphorylation of MAPK. Tissueremodeling in asthmatic airways involves proliferation of smooth musclecells and thickening of basal membranes. It remains to be determinedwhether these pathological changes are influenced by NPSRs endogenouslyexpressed in airway smooth muscle cells.

The C-terminal splice variant of NPSR (NPSR C-alt, GPRA isoform B) wasdescribed to be significantly over-expressed in airway smooth musclecells from asthmatic patients as compared to healthy controls whenstudied by immunohistochemical staining. Our data provide no evidencefor an altered second messenger response elicited by NPSR C-alt ascompared to NPSR WT. It remains to be determined whether the alternativeC-terminal tail of the receptor protein can affect other signalingpathways and thus have functional significance in the pathology ofasthma. However, it seems reasonable to assume that mere over-expressionof NPSR C-alt in airway smooth muscle cells well be sufficient toincrease NPS signaling and thus lead to enhanced bronchial constrictionor tissue remodeling.

At present, the functional involvement of NPSR in airway smooth musclecontraction still remains to be verified. It should also be noted that arecent study failed to detect genetic association of another polymorphicsite in the NPSR/GPRA locus on human chromosome 7 (SNP522363 G>C) inKorean patients. Although this polymorphism is located in an intron anddistant from the SNP producing the Asn¹⁰⁷Ile variant (SNP591694 A>T), itwas found strongly associated with the risk haplotypes in the originalstudy. Two more recent studies investigating large outbred Europeanpopulations seem to confirm that SNP522363 is not associated withincreased risk of asthma while lending support for the overallobservation that specific NPSR haplotypes confer an increased risk ofdeveloping asthma. Therefore, further investigations into the functionalrole of NPSR in asthmatic and healthy lung will be necessary todetermine whether the gain-of-function mutation in NPSR Ile¹⁰⁷ that wedescribe in this paper is involved in the pathological events underlyingasthma. Ultimately the development of NPS antagonists will be a criticalstep to clarify the contribution of NPS signaling in bronchialconstriction. It should also be noted that rat and mice NPSR genes bothcode for an Ile residue at the corresponding position and do not possessthe alternatively spliced exon giving rise to NPSR C-alt. It maytherefore be difficult to study the physiological functions of humanNPSR variants in these rodent model organisms.

Applicants' data show that NPSR can couple to intracellular Ca²⁺ as wellas cAMP pathways, indicating interaction with both G_(q) and G_(s) typesof G proteins. The pharmacophore of NPS is contained within theN-terminal part of the peptide and we describe NPS 1-10 as a minimallyactive structure. All NPS fragments used in our studies could beproduced by proteolytic processing involving trypsin-like cleavage atbasic amino acid residues. Some of these fragments retain potent agonistactivity. Therefore, it will be important to determine the enzymaticsteps involved in the inactivation of this neuropeptide in vivo.Apparently the NPSR protein cannot be studied easily in transienttransfection systems which made the pharmacological analysis of thereceptor variants more tedious. One common problem of using stableclones for second messenger assays is caused by the fact that each clonedisplays an individual pharmacology and comparison of too few stableclones can lead to inaccurate assumptions about general pharmacologicalproperties. Therefore, a large population of stable clones were analyzedand mean EC₅₀ values were determined, followed by statistical analysis.This procedure allowed us to detect significant differences inagonist-induced second messenger coupling between NPSR WT and NPSRIle¹⁰⁷.

Naturally occurring mutations that affect receptor function have beenidentified in numerous GPCRs. Not surprisingly, most of these mutationslead to inactive receptor proteins. The few examples of gain-of-functionmutations can be divided into two classes based on their pharmacologicalphenotype: One group of mutations produces constitutively activereceptors that promote second messenger signaling in the absence ofendogenous agonist. This type of activating mutations has been found inthe glycoprotein-hormone receptor subfamily (LH and TSH receptor),parathyroid and parathyroid-related peptide receptor, as well as inrhodopsin. The other type of mutations increases ligand affinity oragonist efficacy in a way similar to the NPSR Ile¹⁰⁷ variant. Suchmutations have been found in the Ca²⁺-sensing receptor and result inhypocalcemia and hypercalciuria.

Genetic variations in several GPCRs have also been associated withasthma susceptibility or effectiveness of asthma pharmacotherapy. Acoding polymorphism in the cysteinyl-leukotriene receptor type 2(CysLT2) was found to reduce the receptor's affinity to one of its majorendogenous ligands, leukotriene D₄ (LTD₄). Since LTD₄ is an importantmediator of inflammatory responses in asthma this polymorphism in CysLT2provides an asthma-protective effect. Similarly, particular haplotypesin the promotor region of the prostanoid DP receptor (PTGDR) were foundunderrepresented in asthmatic patients. These polymorphisms lead to areduced transcription of the PTGDR mRNA and thus lower levels ofreceptor protein. Prostaglandin D2 (the endogenous ligand of PTGDR) isan important mediator of asthma and PTGDR was found to be required forthe development of airway sensitization in a mouse model of asthma. Thisexplains why reduced levels of PTGDR expression lead to an overallasthma-protective effect. Coding polymorphisms in the β₂-adrenoreceptorthat influence receptor downregulation in response to adrenergicagonists were found to be associated with the therapeutic benefit ofβ₂-agonists to treat symptoms of asthma. One study described thatpatients homozygous for a particular genotype (Arg16/Arg16) ofβ₂-adrenergic receptors displayed a worsening of bronchial airflow underrepeated salbutamol treatment while carriers of the Gly16/Gly16 allelimproved. Although β₂-adrenoreceptors are not causally involved in thepathophysiology of asthma, they are the prime therapeutic targets foracute and intermittent treatment of asthma symptoms. These examplesillustrate the important contribution of specific GPCR genotypes forasthma susceptibility or therapy.

The observation of enhanced NPS-induced second messenger responses atNPSR Ile¹⁰⁷ could also have important consequences for brain functionsince the predominant sites of NPSR expression are found in the centralnervous system. It might be possible that the Ile¹⁰⁷ isoform of NPSR isassociated with changes in behavior or neuronal processing. In summary,we provide evidence that a naturally occurring polymorphism in the NPSRis producing a gain-of-function, resulting in enhanced second messengersignaling that could influence bronchial contractility and tissueremodeling. This polymorphism has been associated with an increased riskof asthma and our present pharmacological data may offer a functionalexplanation how the mutated NPSR protein could contribute topathophysiological changes in asthma.

In some embodiments of the invention, exogenous NPSR agonist (e.g., apreparation comprising isolated NPS) or NPSR antagonist (e.g., apreparation comprising a compound of General Formula I above) may beadministered to a human or animal subject to bring about a desiredtreatment or effect. In other embodiments of the invention,pharmacologic, electrical, radiofrequency, photonic or other means maybe used to cause the subject's body to synthesize or retain increased orelevated amounts of a naturally occurring NPSR agonist or NPSRantagonist to bring about a desired treatment or effect. For example, insome applications, a stimulator or one or more electrodes may beimplanted or positioned in the LC and used to stimulate the productionof endogenous NPS by the LC, thereby augmenting the amounts of NPS thatwould naturally be present in the subject's body without suchstimulation of the LC.

It is to be appreciated that the invention has been described hereabovewith reference to certain examples or embodiments of the invention butthat various additions, deletions, alterations and modifications may bemade to those examples and embodiments without departing from theintended spirit and scope of the invention. For example, any element orattribute of one embodiment or example may be incorporated into or usedwith another embodiment or example, unless to do so would render theembodiment or example unsuitable for its intended use. All reasonableadditions, deletions, modifications and alterations are to be consideredequivalents of the described examples and embodiments and are to beincluded within the scope of the following claims.

What is claimed is:
 1. A method for treating narcolepsy, hypersomnia, oranxiety in a human subject who suffers from such disorder, said methodcomprising the step of: A. administering an NPSR agonist to the subjectin an amount and by a route of administration that is effective to treatsaid disorder; wherein the NPSR agonist comprises NPS or isolated NPS.2. A method according to claim 1 wherein the NPSR agonist comprisesisolated NPS.
 3. A method according to claim 1 wherein the NPSR agonistcomprises NPS which comprises the amino acid sequenceSer-Phe-Arg-Asn-Gly-Val-Gly-Thr-Gly-Met-Lys-Lys-Thr-Ser-Phe-Gln-Arg-Ala-Lys-Ser-OH(SEQ ID NO:1).